Pier Jr Morin

1H NMR metabolomics analysis of glioblastoma subtypes: correlation between metabolomics and gene expression characteristics

Background: Unpredictable clinical behavior of glioblastoma multiforme suggests distinct molecular subtypes. Results: Metabolic profiles of different glioblastoma lines indicate distinct subtypes correlated with gene expression differences. Conclusion: A subset of metabolites can be used to distinguish between four subtypes of glioblastomas. Significance: Metabolic profiling of cancers provides a way for subtype determination with possible diagnostic and prognostic applications. Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by unpredictable clinical behaviors that suggest distinct molecular subtypes. With the tumor metabolic phenotype being one of the hallmarks of cancer, we have set upon to investigate whether GBMs show differences in their metabolic profiles. 1H NMR analysis was performed on metabolite extracts from a selection of nine glioblastoma cell lines. Analysis was performed directly on spectral data and on relative concentrations of metabolites obtained from spectra using a multivariate regression method developed in this work. Both qualitative and quantitative sample clustering have shown that cell lines can be divided into four groups for which the most significantly different metabolites have been determined. Analysis shows that some of the major cancer metabolic markers (such as choline, lactate, and glutamine) have significantly dissimilar concentrations in different GBM groups. The obtained lists of metabolic markers for subgroups were correlated with gene expression data for the same cell lines. Metabolic analysis generally agrees with gene expression measurements, and in several cases, we have shown in detail how the metabolic results can be correlated with the analysis of gene expression. Combined gene expression and metabolomics analysis have shown differential expression of transporters of metabolic markers in these cells as well as some of the major metabolic pathways leading to accumulation of metabolites. Obtained lists of marker metabolites can be leveraged for subtype determination in glioblastomas.

Differential expression of microRNA species in a freeze tolerant insect, Eurosta solidaginis.

Freeze tolerance in insects is associated with a variety of adaptations including production of cryoprotectants, specialized proteins that regulate ice formation, and energy-saving mechanisms that strongly suppress the rates of metabolic processes in the oxygen-limited frozen state. We hypothesized that microRNAs (miRNAs), small non-coding transcripts that bind to mRNA, could play a role in the global regulation of energy-expensive mRNA translation in frozen insects and would be modulated at subzero temperatures. Expression levels of miRNA species were evaluated in control (5 °C) and frozen (-15 °C) goldenrod gall fly larvae, Eurosta solidaginis, using a miRNA microarray. Levels of miR-11, miR-276, miR-71, miR-3742, miR-277-3p, miR-2543b and miR-34 were significantly reduced in frozen larvae whereas miR-284, miR-3791-5p and miR-92c-3p rose significantly in frozen larvae. Target prediction for two miRNAs, miR-277-3p and miR-284, revealed potential regulation of transcripts involved in translation and the Krebs cycle. These data constitute the first report that differential expression of miRNAs occurs in a freeze tolerant insect and suggest a mechanism for reversible gene regulation during prolonged periods of freezing over the winter months, a mechanism that can be rapidly reversed to allow renewed translation of mRNA when temperatures rise and insects thaw.

Differential expression of miRNAs with metabolic implications in hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

Mammalian hibernators undergo significant physiological and biochemical changes when confronted with cold temperatures. Metabolic depression and translational repression are two examples of the various processes impacted during a torpor bout. MicroRNAs (miRNAs), non-coding transcripts that bind to mRNAs, are known regulators of mRNA translation and a growing number of these molecules have been found to be differentially expressed during hibernation. We hypothesized that a group of six miRNAs, with targets involved in various metabolic cascades, is modulated in selected tissues of the hibernating thirteen-lined ground squirrel Ictidomys tridecemlineatus. Expression levels of these miRNAs were assessed in the liver, heart, and skeletal muscle ground squirrel tissues using qRT-PCR. miR-29a, miR-152, miR-195, miR-223, and miR-486 were shown to be up-regulated in the hibernating liver, while miR-378 was shown to be down-regulated in hibernating skeletal muscle tissue samples. Interestingly, fatty acid synthase (FAS), an enzyme involved in fatty acid biosynthesis and a miR-195 target, was shown to be down-regulated in hibernating squirrel liver. This data add to the growing signature of differentially expressed miRNAs during hibernation and puts the light on the potential regulation of fatty acid homeostasis by a miRNA in torpid animals.

NMR Metabolomics Analysis of the Effects of 5-Lipoxygenase Inhibitors on Metabolism in Glioblastomas

Changes across metabolic networks are emerging as an integral part of cancer development and progression. Increasing comprehension of the importance of metabolic processes as well as metabolites in cancer is stimulating exploration of novel, targeted treatment options. Arachidonic acid (AA) is a major component of phospholipids. Through the cascade catalyzed by cyclooxygenases and lipoxygenases, AA is also a precursor to cellular signaling molecules as well as molecules associated with a variety of diseases including cancer. 5-Lipoxygenase catalyzes the transformation of AA into leukotrienes (LT), important mediators of inflammation. High-throughput analysis of metabolic profiles was used to investigate the response of glioblastoma cell lines to treatment with 5-lipoxygenase inhibitors. Metabolic profiling of cells following drug treatment provides valuable information about the response and metabolic alterations induced by the drug action and give an indication of both on-target and off-target effects of drugs. Four different 5-lipoxygenase inhibitors and antioxidants were tested including zileuton, caffeic acid, and its analogues caffeic acid phenethyl ester and caffeic acid cyclohexethyl ester. A NMR approach identified metabolic signatures resulting from application of these compounds to glioblastoma cell lines, and metabolic data were used to develop a better understanding of the mode of action of these inhibitors.

PF-8380 and closely related analogs: synthesis and structure-activity relationship towards autotaxin inhibition and glioma cell viability.

A series of PF‐8380 analogs, a recently developed autotaxin inhibitor, was explored. Inhibition of autotaxin by these analogs, as well as by all PF‐8380 synthetic intermediates, shows the importance of meta‐dichlorobenzyl and benzo[d]oxazol‐2(3H)‐one fragments. However, analogs 8 and 9, bearing only the benzo[d]oxazol‐2(3H)‐one moiety, are more cytotoxic on the LN229 glioblastoma cell line than PF‐8380 and temozolomide (TMZ).

Investigating a signature of temozolomide resistance in GBM cell lines using metabolomics

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Current therapeutic approach to treat this malignancy involves a combination of surgery, radiotherapy and chemotherapy with temozolomide. Numerous mechanisms contributing to inherent and acquired resistance to this chemotherapeutic agent have been identified and can lead to treatment failure. This study undertook a metabolomics-based approach to characterize the metabolic profiles observed in temozolomide-sensitive and temozolomide-resistant GBM cell lines as well as in a small sub-set of primary GBM tumors. This approach was also utilized to explore the metabolic changes modulated upon cell treatment with temozolomide and lomeguatrib, an MGMT inhibitor with temozolomide-sensitizing potential. Metabolites previously explored for their potential role in chemoresistance including glucose, citrate and isocitrate demonstrated elevated levels in temozolomide-resistant GBM cells. In addition, a signature of metabolites comprising alanine, choline, creatine and phosphorylcholine was identified as up-regulated in sensitive GBM cell line across different treatments. These results present the metabolic profiles associated with temozolomide response in selected GBM models and propose interesting leads that could be leveraged for the development of therapeutic or diagnostic tools to impact temozolomide response in GBMs.

Expression of miRNAs in response to freezing and anoxia stresses in the freeze tolerant fly Eurosta solidaginis.

Insect cold hardiness is associated with substantial metabolic rate suppression, often including developmental diapause as well as metabolic suppression imposed by freezing and freeze-associated oxygen limitation. MicroRNAs, small non-coding transcripts that bind to mRNA, are known modulators of hypometabolism in freeze tolerant insects. To further contribute to the growing signature of stress-responsive miRNAs, this study amplified and quantified changes in the expression levels of four microRNA species, miR-8, miR-9, miR-92b and miR-277, in response to freezing or anoxia exposures of freeze tolerant gall fly larvae, Eurosta solidaginis. MiR-92b levels were significantly elevated by 1.57-fold in frozen E. solidaginis at -15°C as compared with 5°C controls, whereas miR-92b levels were significantly reduced in anoxic E. solidaginis to levels that were 0.77-fold as compared with larvae held under normoxic conditions. The other miRNAs investigated showed no significant changes in stressed larvae. These data demonstrate differential miR-92b expression in frozen/anoxic versus control insect larvae and position this miRNA as a stress responsive marker in this model insect.

miRNAs as important drivers of glioblastomas: A no-brainer?

There is no debate on the relevance of miRNAs in the pathogenesis of cancer. Numerous miRNAs with oncogenic and tumor-suppressive properties have been identified in glioblastoma multiforme (GBM), an aggressive type of brain tumor with dismal prognosis. Differential expression of these biomolecules in several cancer models makes them attractive therapeutic targets for the development of miRNA-based cancer treatments despite the hurdles associated with such an approach. In addition, systemic release of miRNAs also positions them as attractive tools for non-invasive cancer diagnosis and prognosis. This review initially looks at differentially expressed miRNAs in GBMs. Our focus will next be directed towards circulating miRNAs and how these molecules could be leveraged for cancer diagnosis as well as for the assessment of patient response to chemotherapeutic treatments. Finally, we discuss the primary strategies utilized in the development of miRNA-focused therapeutics and summarize preclinical results gathered in GBMs to date.

Metabolic Effects of Known and Novel HDAC and SIRT Inhibitors in Glioblastomas Independently or Combined with Temozolomide

Inhibition of protein deacetylation enzymes, alone or in combination with standard chemotherapies, is an exciting addition to cancer therapy. We have investigated the effect of deacetylase inhibition on the metabolism of glioblastoma cells. 1H NMR metabolomics analysis was used to determine the major metabolic changes following treatment of two distinct glioblastoma cell lines, U373 and LN229, with five different histone deacetylase (HDAC) inhibitors, as well as one inhibitor of NAD+-dependent protein deacetylases (SIRT). The addition of the standard glioblastoma chemotherapy agent, temozolomide, to the HDAC and SIRT treatments led to a reduction in cell survival, suggesting a possibility for combined treatment. This study shows that distinct glioblastoma cell lines, with different metabolic profiles and gene expression, experience dissimilar changes following treatment with protein deacetylase inhibitors. The observed effects of inhibitors on mitochondrial metabolism, glycolysis and fatty acid synthesis suggest possible roles of protein deacetylases in metabolism regulation. Metabolic markers of the effectiveness of anti-protein deacetylase treatments have been explored. In addition to known deacetylation inhibitors, three novel inhibitors have been introduced and tested. Finally, 1H NMR analysis of cellular metabolism is shown to be a fast, inexpensive method for testing drug effects.

Characterization of cold-associated microRNAs in the freeze-tolerant gall fly Eurosta solidaginis using high-throughput sequencing

Significant physiological and biochemical changes are observed in freeze-tolerant insects when confronted with cold temperatures. These insects have adapted to winter by retreating into a hypometabolic state of diapause and implementing cryoprotective mechanisms that allow them to survive whole body freezing. MicroRNAs (miRNAs), a family of short ribonucleic acids, are emerging as likely molecular players underlying the process of cold adaptation. Unfortunately, the data is sparse concerning the signature of miRNAs that are modulated following cold exposure in the freeze-tolerant goldenrod gall fly Eurosta solidaginis. Leveraging for the first time a next-generation sequencing approach, differentially expressed miRNAs were evaluated in 5°C and -15°C-exposed E. solidaginis larvae. Next-generation sequencing expression data was subsequently validated by qRT-PCR for selected miRNA targets. Results demonstrate 24 differentially expressed freeze-responsive miRNAs. Notable, miR-1-3p, a miRNA modulated at low temperature in another cold-hardy insect, and miR-14-3p, a miRNA associated with stress response in the fruit fly, were shown to be significantly up-regulated in -15°C-exposed larvae. Overall, this work identifies, for the first time in a high-throughput manner, differentially expressed miRNAs in cold-exposed E. solidaginis larvae and further clarifies an emerging signature of miRNAs modulated at low temperatures in cold-hardy insects.